With advent of point-of-care (POS) measurements, the importance of, and demand for, gene analysis, in vitro diagnosis (IVD), and gene base sequence analysis continue to increase. To meet the growing importance and demand, platforms and systems have been developed to perform a large number of tests with a small amount of samples. For example, microfluidic platforms and systems for microfluidic chips or lab-on-a-chip applications are receiving considerable attention for high throughout analysis. A microfluidic device includes a plurality of microfluidic channels and chambers designed to control and manipulate a very small amount of fluid. Using a microfluidic device may minimize the reaction time of microfluid and allow simultaneously the reaction of microfluid and measurement of the reaction result. Such a microfluidic device may be fabricated using various methods in which diverse materials are used.
Meanwhile, for example, during gene analysis, in order to precisely detect the presence of specific DNA or the amount of the DNA in a sample, an actual sample has to be sufficiently amplified for measurement after its purification/extraction. Among various gene amplification techniques, polymerase chain reaction (PCR) amplification is most widely used. A fluorescence detection method is commonly used to detect DNA amplified through PCR. For example, quantitative real-time PCR (qPCR) uses a plurality of fluorescent dyes/probes and primer sets to amplify and detect/measure a target sample in real time. The principle of qPCR using a TaqMan probe is that a TaqMan probe falls off a template during DNA amplification, thereby having fluorescent characteristics. That is, as a PCR cycle progresses, the number of TaqMan probes cleaved from each template exponentially increases. As a result, the level of a fluorescence signal exponentially increases. By measuring a change in such a fluorescence signal level with an optical system, the presence of a target sample may be determined and a quantitative analysis of the target sample may be allowed. As a PCR cycle progresses, a fluorescence signal level follows an S-curve in which a threshold cycle Ct is set and measured at the point where the fluorescence signal level rapidly changes. Platforms for IVD, gene analysis, biomarker development, gene base sequence analysis using a qPCR technique have already been commercially available.
A fluorescence detecting optical system is configured to measure the level of a fluorescence signal or a change in the level thereof due to bio reactions within a microfluidic device such as a microfluidic chip or PCR chip. For example, a fluorescence detecting optical system may irradiate excitation light on a sample labeled with a fluorescent dye and detect fluorescence radiated from the fluorescent dye excited with the excitation light. However, most fluorescent dyes have a wavelength region overlapping between wavelength bands of excitation light and fluorescent light. Thus, an excitation light filter and a fluorescent light filter may be designed in such a way that a wavelength band passed by the excitation light filter does not overlap a wavelength band passed by the fluorescent light filter. If the wavelength bands passed by the excitation light filter and the fluorescent light filter overlap each other, the excitation light is not completely separated from the fluorescent light, thereby causing the excitation light reflected from the microfluidic device to be incident on a photodetector. Because excitation light is usually about 105 to 106 times brighter than fluorescent light emitted by a fluorescent dye, crosstalk between the excitation light and the fluorescent light may degrade the detection performance of a fluorescence detecting optical system.
A multi-channel fluorescence detection apparatus including a plurality of fluorescence detecting optical systems for detecting different colors of fluorescence may suffer from crosstalk between excitation light in adjacent wavelength ranges and between fluorescent light in adjacent wavelength ranges according to the design of an excitation light filter, a fluorescent light filter, and a dichroic filter because there is an overlapping region between excitation light in adjacent wavelength ranges and between fluorescent light in adjacent wavelength ranges.
In order to prevent occurrence of such crosstalk, the excitation light filter and the fluorescent light filter are usually designed to pass a narrow wavelength band (several tends of nm). However, as wavelength ranges transmitted by the excitation light filter and the fluorescent light filter decrease, the intensities of the excitation light and the fluorescent light decreases, thereby resulting in the degradation of detection performance. Furthermore, it is difficult to design a plurality of excitation light filters and a plurality of fluorescent light filters so that transmission wavelength bands thereof do not overlap each other. In order to overcome the drawbacks, compensation of crosstalk using software has been proposed. However, this approach is difficult to apply because the result may vary greatly depending on a selected coefficient value.
Furthermore, a portion of excitation light emitted by a light source in a fluorescence detecting optical system is reflected back from a microfluidic device to the light source. This may cause an interference between excitation light emitted by the light source and that reflected from the microfluidic device. The interference between the excitation light may result in noise, thereby adversely affecting the detection performance of the fluorescence detecting optical system.